Study Of Ctx-m-14 Extended Spectrum β-lactamases In Producing Esbls Escherichia Coli

Background and Objective:The producing of extended-spectrumβ-lactamases(ESBLs) is the most common mechanism of resistance to the third generation cephalosporins in Enterobacteriaceae bacteria,especially Escherichia coli and Klebsiella pneumoniae.ESBLs are mostly mediated by plasmid,which can hydrolyze oxyimineolactam antibiotics and Helicesβ-lactamases antibiotics,which can be inhibited byβ-lactamases inhibitors. Nowadays,hundreds of species of ESBLs had been discovered,including TEM,SHV, CTX-M,OXA and other type etc.Plasmid carrying ESBLs genes are easily transmitted among different members of Enterobacteriaceae.It is limited for clinical anti-infection treatment to choose antibiotics against the multiple-drug resistant bacteria.At present,there have been a lot of researches about subtypes of ESBLs in U.S.A, France.etc.It is different in the epidemic and antibiotic susceptibilities of ESBLs, because of areas,environment,medical level and usage of antibiotics.Some investigators found,CTX-M ESBLs is predominant genotype of ESBLs in China and CTX-M-14 ESBLs is the most important subtype.In order to study the effect of CTX-M-14 ESBLs in epidemic and antibiotic susceptibilities in He Nan province,we investigated the prevalence and resistance of CTX-M-14 ESBLs producers in E.coli. Methods:1.156 strains ofEscherichia coli were collected from the First Affiliated Hospital of Zhengzhou University,from Oct 2006 to Oct 2007.Those clinical isolates were identified by Two-paper cooperate test and E-test.Then collective plasmids were extracted and amplified by PCR using primer of CTX-M-14 and collect the positive fragments.2.the positive fragments were digested by BamH I and Xho I,they were cloned into pET28(a+) vector and transformed to E.coli BL-21 sensitive cells.Then we extracted the re-combined plasmid and inspected its nucleotide sequence to prove the objective gene CTX-M-14.3.Use Isopropyl-β-D-thiogalactoside(IPTG) to induce the expressing of CTX-M-14 gene.After the inclusion bodies were isolated,they were purified by dialysis and re-natured by urea of low concentration.Judge their biological activity in Mueller-Hinton plate.4.We detected antibiotic susceptibilities of the transformant by agar dilution test.Results:1.Among the 156 ESBLs positive strsins,we obtained 67 strains of the CTX-M-14, the positive rate is 42.9 percentage.The positive fragment is between 750 and 1000bp.2.After the PCR products were cloned to pET28(a+) vetor and transformed to BL-21 cells,we extracted the recombinant plasmid and cut it by the same enzyme, and the DNA fragment the same as PCR product was found,which was identified with the CTX-M-14 gene sequence in Genebank.3.In analyzing the protein activity,SDS-PAGE show that the objective fragment is between 27.0KD and 30.0KD,which accord with CTX-M-14 28KD.4.Transformant was resistant to penicillins,the first and second generation cephalosporins,cefotaxime and ceftriaxone,and it was sensitive to tetracycline, gentamicin,aminoglycosides,imipenem,transformant was also sensitive to antibiotics including beta-lactamase inhibitors and Ceftazidime in vitro. Conclusion:This study has shown that high prevalence of CTX-M-14 ESBLs producers is observed in Escherichia coli in HeNan province,this type is predominant genotype of ESBLs in our district.It's easy to succeed by dialyzing to purify the protein and by depending on low concentration of uric to re-nature it.The transformant possess wide resistance to antibiotics and the resistant spectrums of transformant were similar to the investigation in our country.It is upmost importance to monitor,detect CTX-M-14 ESBLs-producing strains closely,control and prevent it spread.